Ivanka Karadzic

Purification and characterization of a protease from Pseudomonas aeruginosa grown in cutting oil

The Pseudomonas aeruginosa san-ai strain was isolated from water-soluble cutting oil used for cooling and lubrication during industrial metal-working processes. This strain, which is grown in a high alkaline (pH 10) mixture of surfactants and mineral oil, produces an extracellular proteolytic enzyme. We have purified and characterized this 18 kDa protease. The P. aeruginosa san-ai protease functions optimally at pH 9.0 and 60 degrees C. Additionally, it is a Zn-containing metalloenzyme, and its monomeric structure contains at least one disulfide bond. Because the enzyme is stable in the presence of organic solvents, it is suitable for peptide synthesis. Furthermore, the P. aeruginosa san-ai protease could be used in an intelligent drug delivery system (DDS) designed for applications in the metal industry for prevention of putrefaction of cutting oil.

Genetic and Proteomic Analyses of a Proteasome-Activating Nucleotidase A Mutant of the Haloarchaeon Haloferax volcanii

The halophilic archaeon Haloferax volcanii encodes two related proteasome-activating nucleotidase proteins, PanA and PanB, with PanA levels predominant during all phases of growth. In this study, an isogenic panA mutant strain of H. volcanii was generated. The growth rate and cell yield of this mutant strain were lower than those of its parent and plasmid-complemented derivatives. In addition, a consistent and discernible 2.1-fold increase in the number of phosphorylated proteins was detected when the panA gene was disrupted, based on phosphospecific fluorescent staining of proteins separated by 2-dimensional gel electrophoresis. Subsequent enrichment of phosphoproteins by immobilized metal ion and metal oxide affinity chromatography (in parallel and sequentially) followed by tandem mass spectrometry was employed to identify key differences in the proteomes of these strains as well as to add to the restricted numbers of known phosphoproteins within the Archaea. In total, 625 proteins (approximately 15% of the deduced proteome) and 9 phosphosites were identified by these approaches, and 31% (195) of the proteins were identified by multiple phosphoanalytical methods. In agreement with the phosphostaining results, the number of identified proteins that were reproducibly exclusive or notably more abundant in one strain was nearly twofold greater for the panA mutant than for the parental strain. Enriched proteins exclusive to or more abundant in the panA mutant (versus the wild type) included cell division (FtsZ, Cdc48), dihydroxyacetone kinase-linked phosphoenolpyruvate phosphotransferase system (EI, DhaK), and oxidoreductase homologs. Differences in transcriptional regulation and signal transduction proteins were also observed, including those differences (e.g., OsmC and BolA) which suggest that proteasome deficiency caused an up-regulation of stress responses (e.g., OsmC versus BolA). Consistent with this, components of the Fe-S cluster assembly, protein-folding, DNA binding and repair, oxidative and osmotic stress, phosphorus assimilation, and polyphosphate synthesis systems were enriched and identified as unique to the panA mutant. The cumulative proteomic data not only furthered our understanding of the archaeal proteasome system but also facilitated the assembly of the first subproteome map of H. volcanii.

Proteasomes: perspectives from the Archaea.

The development of whole systems approaches to microbiology (e.g. genomics and proteomics) has facilitated a global view of archaeal physiology. Surprisingly, as archaea respond to environmental signals, the majority of protein concentration changes that occur are not reflected at the mRNA level. This incongruity highlights the importance of post-transcription control mechanisms in these organisms. One of the central players in proteolysis is the proteasome, a multicatalytic energy-dependent protease. Proteasomes serve both proteolytic and non-proteolytic roles in protein quality control and in the regulation of cell function. The proteolytic active sites of these enzymes are housed within a central chamber of an elaborate nanocompartment termed the 20S proteasome or core particle. Axial gates, positioned at each end of this particle, restrict the type of substrate that can access the proteolytic active sites. Assortments of regulatory AAA complexes are predicted to recognize/bind and unfold substrate proteins, open the axial gates, and translocate substrate into the 20S core particle.

Chemical cross-linking, mass spectrometry, and in silico modeling of proteasomal 20S core particles of the haloarchaeon Haloferax volcanii

A fast and accurate method is reported to generate distance constraints between juxtaposited amino acids and to validate molecular models of halophilic protein complexes. Proteasomal 20S core particles (CPs) from the haloarchaeon Haloferax volcanii were used to investigate the quaternary structure of halophilic proteins based on their symmetrical, yet distinct subunit composition. Proteasomal CPs are cylindrical barrel‐like structures of four‐stacked homoheptameric rings of α‐ and β‐type subunits organized in α7β7β7α7 stoichiometry. The CPs of H. volcanii are formed from a single type of β subunit associated with α1 and/or α2 subunits. Tandem affinity chromatography and new genetic constructs were used to separately isolate α17β7β7α17 and α27β7β7α27 CPs from H. volcanii. Chemically cross‐linked peptides of the H. volcanii CPs were analyzed by high‐performance mass spectrometry and an open modification search strategy to first generate and then to interpret the resulting tandem mass spectrometric data. Distance constraints obtained by chemical cross‐linking mass spectrometry, together with the available structural data of nonhalophilic CPs, facilitated the selection of accurate models of H. volcanii proteasomal CPs composed of α1‐, α2‐, and β‐homoheptameric rings from several different possible structures from Protein Data Bank.

Leucine Aminopeptidase from Streptomyces hygroscopicus is Controlled by a Low Molecular Weight Inhibitor

In culture filtrate of Streptomyces hygroscopicus a producer of polyketide antibiotics, a leucine aminopeptidase and its autogenous inhibitor were detected. The leucine aminopeptidase was purified 4573-fold with yield of 82% by combination of ion exchange and hydrophobic chromatography. The enzyme is monomeric with a molecular mass of 51 kDa determined by gel chromatography and 67 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimal activity was at pH 8.0 and 40 degrees C. The pI of leucine aminopeptidase is 8.2. The enzyme is strongly inhibited by 1,10-phenantroline, amastatin and dithiothreitol. Atomic absorption spectrometry indicated 2 mols of ion zinc per mol of enzyme. The enzyme is stable at up to 70 degrees C. Leucine aminopeptidase prefers leucine and methionine as N-terminal amino acids. Activity of leucine aminopeptidase is strongly modulated by an autogenous low-molecular weight inhibitor during fermentation, especially during periods of intensive antibiotic production.

Enzymatic characterization of 30 kDa lipase from Pseudomonas aeruginosa ATCC 27853

An extracellular lipase from Pseudomonas aeruginosa ATCC 27853 has been purified and its enzymatic characteristics were determined. According to SDS‐PAGE and gel filtration molecular mass estimated to be 30 kDa, what classified the lipase in group I.1. Although 14 lipases from P. aeruginosa with similar molecular mass are referred to date, their basic enzymatic properties have not been reported yet. To address the gap we found: the optimal temperature and pH in water solution being 50 °C and 9.3, respectively; the lipase was inhibited with Hg2+ ions and sodium dodecylsulphate (SDS), while non‐ionic detergent Triton X‐100 activated the enzyme; the lipase hydrolyzed more rapidly middle chain triglycerides and it was not regiospecific; the lipase demonstrated naturally occurring stability in different organic solvents with concentrations ranging from 30 to 70%, including good thermal stability in 30% organic solvent solution. Even though strain P. aeruginosa ATCC 27853 was not isolated from extreme environment it showed activity in organic solvent suggesting that this lipase is suitable for variety of applications, including reactions in water restricted medium and bioremediation of contaminations by organic solvents. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Structure‐Function Relationships of Rhamnolipid and Exopolysacharide Biosurfactants of Pseudomonas aeruginosa as Therapeutic Targets in Cystic Fibrosis Lung Infections

Chronic Pseudomonas aeruginosa lung infection is the cause of much morbidity and most of the mortality in cystic fibrosis (CF) patients. The high prevalence of P. aeruginosa infec‐ tions in CF is related to the microbes large genome and mechanisms of adaptation to the CF lung environment, the host immune system and antibiotic resistance. Among a wide range of P. aeruginosa metabolites involved in infection development in CF, the biosurfactant compounds, rhamnolipids (RLs) and exopolysaccharides (EPSs), have important roles in the early stages of P. aeruginosa infection in CF. RLs and EPSs are involved in bacterial adhesion, biofilm formation, antibiotic resistance, and impairment of host immune system pathways, as well as in processes such as biofilm maintenance and the mucoid phenotype of P. aeruginosa, which lead to development of chronic infec‐ tion. Due to the proposed roles of RLs and EPSs and the importance of prevention and treatment of P. aeruginosa respiratory infections in CF, these compounds are promising targets for patient therapy. In the future, impairment of P. aeruginosa quorum sensing (QS) pathways and modification of host respiratory mucus epithelial membranes should be considered as potential approaches in preventing respiratory infections caused by this microbe in CF patients.